PROJECT ABSTRACT Infections of the central nervous system (CNS) caused by fungi have the highest morbidity and mortality when compared to other causative agents of CNS infections. Cryptococcus neoformans (Cn), Candida albicans, Aspergillus spp., Mucor, Coccidioides spp., and Histoplasma capsulatum are the most common cause of fungal brain infections resulting in a chronic instead of an acute or subacute meningoencephalitis. The long-term goal will investigate the spatio-temporal organization and molecular basis of Cn trafficking across the BBB and investigate a cross-talk between a transcellular mechanism and the opening of a paracellular path in response to Cn infection. Given our preliminary data and published studies, we propose that Cn represents an excellent model pathogen with which to determine the mechanisms that pathogenic fungi use to breach the BBB and enter the CNS. In our efforts to understand the molecular basis mediating the Cn-brain endothelium interactions, we identified a tyrosine kinase receptor (EphA2-TKR) as the central player. The central hypothesis states that fungal cells penetrate the BBB by engaging EphA2-TKR in order to access a binary path into the CNS. To test our hypothesis, we will investigate the interplay between fungal cells, EphA2-TRK and its downstream signaling components in mediating the translocation of Cn from blood-to-brain. The following specific aims will test the hypothesis: SA1 will investigate a Cn-induced CD44-mediated transactivation of EphA2-TKR activity and the interactome of EphA2-TKR. SA2 will examine the role of EphA2-TKR in upregulating vesicular traffic of Cn and resolve whether Cn-induced activity of EphA2-TKR promotes a co-regulation of a transcellular and paracellular pathway. SA3 will resolve the contribution of EphA2-TKR activity to the translocation of Cn from blood-to-brain in vivo and examine the molecular basis of changes in the permeability of the BBB when challenged by Cn. The proposed research is innovative because it will for the first time, detail the key players that promote the internalization and trafficking of fungi across the BBB. The innovation extends to our published studies that identified EphA2-TKR in the brain endothelium as a central element in mediating the migration Cn from blood-to-brain. This along with the notion that crossing of the BBB is initiated by a fungal-induced CD44-transactivation of EphA2-TKR to trigger a pathological use of endocytosis (and possibly a secondary paracellular path) in brain endothelial cells is a paradigm shift from our current understanding. We have proposed experiments that will use multiple complementary approaches to tease apart the molecular interaction between Cn, EphA2- TKR and CD44 while also using proximity-dependent labeling to identify the interactome of EphA2-TKR. The knowledge gained from our studies will make a significant contribution to fungal pathogenesis of the CNS, because it remains significantly understudied, and this work will spearhead the development of molecules that could block fungal entry and thus serve as pre- emptive or prophylactic therapy in patients at risk for developing fungal meningoencephalitis.